Monoclonal antibodies have considerable usefulness as diagnostic and therapeutic agents in clinical, commercial and research applications. Refinements of the general technique for hybridoma production developed by Kohler and Milstein in 1975 (Nature 256: 495-497) make it possible to produce large quantities of monoclonal antibodies which are able to recognize specific antigenic determinants.
While development of antibodies reactive to protein antigenic sites is known and repeatable, fabrication of monoclonal antibodies reactive to small organic chemicals, such as carcinogens, pesticides, toxic chemicals and DNA adducts is less straight-forward. Production of antibodies to small organic molecules may sometimes be achieved by first linking a small molecule, which is termed a hapten, to a carrier protein, and the immune reactive cells may respond to an antigenic determinant site of this complex. Initial chemical treatment of the small molecule hapten, may be required in order for it to be conjugated to an immunoreactive carrier protein. The immunoreactive cells are induced to form antibodies which have recognition sites to various reactive sites located (1) on the hapten molecule, (2) the carrier protein, (3) the hapten carrier-protein complex, or (4) any combination of the hapten, the linkage chemistry and the carrier protein. The specific reactive site is not known and is unpredictable. The site and chemistry of the hapten conjugation to the carrier protein may influence the specificity of the antibodies produced.
Antibodies with specific binding to reactive sites on small organic molecules are sensitive indicators, which may be used to distinguish chemical isomers (Stanker et al, Toxicology 45: 229-243 1987). With small haptens, the greatest antibody specificity for a reactive group appears to occur when that reactive group is most distant from the site of the linkage binding to the carrier protein. This work with synthetic pyrethroids demonstrates that conjugation of the hapten antigenic site as far as possible from the 3-phenoxybenzyl group favors the production of antibodies which preferentially recognize the phenoxybenzyl group. The phenoxybenzyl group is shared by many synthetic pyrethroids, and thus antibodies induced against one of the reactive groups of synthetic pyrethroids may recognize several members of the class of synthetic pyrethroids which carry that reactive group.
Synthetic pyrethroids are a large group of insecticides which are widely used in the United States and other countries. The synthetic pyrethroids most commonly used in the United States are permethrin, cypermethrin and deltamethrin; other synthetic pyrethroids include phenothrin, fenpropathrin, flucythranate, fenvalerate, and tetramethrin, among other compounds. The three most commonly used compounds contain both a phenoxybenzyl and a cyclopropane moiety. The other less used synthetic pyrethroids contain at least one of these groups, or a phenoxyphenyl moiety in place of the phenoxybenzyl moiety. In 1982, synthetic pyrethroids represented as much as 30% of the world insecticide market.
The synthetic pyrethroids in use differ widely in their chemical structure, toxicity and photostability. Historically, the agricultural use of synthetic pyrethroids, such as allethrin and bioallethrin, was limited due to the unstable character of those compounds in the air and water. However, recently developed synthetic pyrethroids such as permethrin, cypermethrin and deltamethrin are more stable and thus have greater agricultural utility. These compounds are widely used for insect control in food processing plants because they display low mammalian toxicity. Widespread use of these more stable compounds, however, has led to concern about the possiblity that pesticide residues might remain in foodstuffs and the environment. Both cypermethrin and permethrin have been listed as potentially oncogenic pesticides by the U.S. Environmental Protection Agency. (See Regulating Pesticides in Food: The Delaney Paradox, National Research Council Board on Agriculture, National Academy Press, Washington, D.C., 1987.)
Residue limits in meats and fats have been established in the United States for permethrin, cypermethrin and deltamethrin. Residue limits for many pyrethroids have been set by the Food and Agricultural Organization and the World Health Organization. However, the lack of convenient, rapid detection systems has hampered environmental identification and quantification of pyrethroids. Conventional analysis involves multi-step sample clean-up procedures followed by gas chromatography, and due to thermal instabilities, detection is limited to electron capture (GC/EC). Analysis by high-pressure liquid chromatography (HPLC) has also been described, but this separation technique cannot adequately resolve the various synthetic pyrethroids. Detection is also a problem with HPLC analysis. The complexity of standard chemical extraction and purification of compounds of such low incidence has engendered a need for other means by which to identify, rapidly and specifically, and to quantify these materials. Specific characterization of the presence and concentration of these compounds by assay with anti-pyrethroid monoclonal antibodies would permit, rapid, automatable analysis of these materials in foods and environmental samples.